Mechanism Of Tribenuron-methyl (TM)-induced Male Sterility In Brassica Napus And Role Of Autophagy In TM Resistance

Rapeseed(Brassica napus)is a major oil crops.The increase in rapeseed production over the past few decades is mainly due to the utilization of heterosis.Chemical hybridization agent(CHA)-induced male sterility has been proposed for heterosis utilization.Selective male sterility could be inducted by foliar spraying of TM at low doses.Tribenuron-methyl(TM)is a powerful sulfonylurea herbicide(SU)that inhibits branched-chain amino acid(BCAA)biosynthesis by targeting the catalytic subunit(CSR1)of acetolactate synthase(ALS).Although TM as an effective CHA has been wildly used in hybrid rapeseed production,the underlying mechanism for the selective induction of male sterility by CHA TM remains unknown.Moreover,with the extensive application of SU herbicides in the past decades,plants have evolved target-site resistance(csr1-1D)and metabolic resistance(Bel)for SU herbicides.Herbicide resistance has become a threat to global agriculture,but no novel TM-induced mechanism for SU resistance has been reported.In this study,we uncovered the mechanism of TM-induced male sterility,and attempted to investigate the role and regulatory mechanism of TM-induced autophagy in SU resistance,through multiple analysis methods(including transgenic,genetic,immunoblotting,and physiological-biochemical analyses)in two rapeseed lines and Arabidopsis plants.After TM application,greater TM accumulation and subsequent stronger ALS inhibition and BCAA starvation in anthers than in leaves and stems.Constitutive or anther-specific expression of csr1-1D(a CSR1 mutant that confers SU herbicide resistance)eliminated anther-selective ALS inhibition and reversed the TM-induced male sterile phenotype in both rapeseed and Arabidopsis,suggesting that ALS is the sole target of TM and inactivation of ALS is the primary cause of TM-induced male sterility.The TM daub-stem experiments showed that sterile anthers only occurred on the TM-daubed branches;and when TM was daubed on the main stem,the anthers on the main stem and those on all the branches above the places that were daubed with TM on main stem were sterile,whereas the rest of the branches were fertile.These results indicate that TM is mainly transported upward.Constitutive expression of the TM metabolism gene Bel reversed the anther-specific TM accumulation and male sterility.Little TM accumulation in anthers and reversion of TM-induced male sterility were also observed through targeted expression of Bel in either the mesophyll or phloem,suggested that foliar-sprayed TM was polar-transported to anthers mainly through the mesophyll and phloem.Autophagy(specifically macroautophagy)is a bulk degradation system,during which bulk cytoplasmic contents,such as protein aggregates and organelles,are sequestered into autophagosome and then are delivered to vacuole for degradation.As autophagy is commonly observed in these dying cells,it is classified as autophagic cell death.Microscopy and immunoblotting revealed that autophagy was elevated in TM-induced male sterile anthers and by anther-specific knockdown of ALS.Compared with control,larger vacuoles and more lightly stained cytoplasm were observed in the degraded microspores and tapetum cells of TM-treated plants.Moreover,TM-induced pollen abortion was significantly inhibited by the autophagy inhibitor 3-MA.These data suggested that TM was polar-transported to anthers,resulting in BCAA starvation via anther-specific ALS inhibition and ultimately autophagic cell death in anthers.Previously study confirmed the general control nonderepressible 2(GCN2)pathway also involves in SU resistance.In yeast and mammals,great progress has been achieved in TOR-autophagy and GCN2-mediated amino acid signaling.In this study,we report autophagy participates in SU herbicide tribenuron-methyl(TM)resistance and amino acid signaling in parallel with GCN2 in Arabidopsis.The activation and reversion of autophagy and GCN2 by TM and exogenous BCAA respectively,confirm that TM-induced BCAA starvation is responsible for the activatation of autophagy and GCN2.Compared with wild type seedlings,genetic analyses reveal more sensitive phenotypes in atg5,atg7,and gcn2 single mutants,but lowest and supersensitive of these in atg5 gcn2 and atg7 gcn2 double mutants after TM treatment.Immunoblotting also revealed that TM-induced activation of autophagy and GCN2 are not dependent on the presence of each other.TM causes inactivation of TOR,a sensor and regulator of amino acid availability,meanwhile estradiol-induced inhibition of TOR induces activation of autophagy but not GCN2,suggesting TM-induced BCAA starvation activates autophagy probably via TOR inativation.Furthermore,autophagy and GCN2 are also activated and independently contribute to TM resistance in Bel,not csr1-1D,transgenic plants.Together,these data suggest that autophagy as a proteolytic process for amino acid recycling contributes to GCN2-independent SU resistance probably due to its ability to replenish fresh BCAA.This study reports that inhibition of ALS activity in anther by TM could cause BCAA starvation,which resulted in autophagy in anther cells and led to male sterility.The results could provide a new insight into the mechanism underlying the TM-induced male sterility,and has a great potential application in crop hybrid seed production.On the other hand,we report that SU herbicide TM probably via TOR inhibition to activate autophagy,which degrades proteins and recycle amino acids to replenish fresh BCAA.Thus,autophagy should be considered as a novel mechanism for SU resistance.TM can also serve as a specific inhibitor of BCAA biosynthesis to investigate the molecular mechanisms of amino acid signaling in plants.This study not only reveals the regulation mechanism of autophagy in SU resistance,but also advances the understanding of amino acid signaling in plants.